Alzheimer's disease (AD) like pathology
Introduction
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder which
results in dementia and death. AD pathology is characterized by senile plaques and
neurofibrillary tangles (NFTs), combined with massive neuronal loss, mainly in the
hippocampus and association regions of the neocortex (Ball and Lo, 1977). The major
constituents of senile plaques are 39?43 amino acid peptides (A β), snipped from a larger protein
called A β precursor protein (APP) (Glenner and Wong, 1984; Masters et al., 1985; Goldgaber
et al., 1987). Recent studies indicate that APP is processed by a group of secretases. The α-
secretase generates a soluble product, while β-secretase and γ-secretase generate A β from APP.
The sporadic nature of most AD cases strongly argues for an environmental link that may drive
AD pathogenesis; however, it is not clear when this may occur. Reconstructions of neonatal
and medical histories of birth cohorts have led to the origin of ‘the Barker hypothesis’ (Barker
et al., 1989; Osmond and Barker, 2000), which links early life experiences and adult diseases.
These observations resulted in a new concept regarding certain adult diseases that emphasizes
the role of environmental factors operating during the pre-conceptual, fetal and infantile phases
of life (Gluckman and Hanson, 2004).
The pathological manifestations in AD patients are presumed to result from defects of old age;
however, it is unlikely that the disease process begins late in life. Therefore, amyloidogenesis
associated with AD can also be viewed as a pathological outcome that is evident during aging;
however, the preceding initiating event may have occurred during early stages of brain
development (Zawia and Basha, 2005). Specifically, this event would have been a latent early-
life associated regulation (LEARn) alteration (Lahiri et al., 2007) affecting the expression of
genes associated with a later-manifest condition.
Previous work from our laboratories showed that developmental exposure of rats to the metal
Pb from birth to PND20 showed a delayed over-expression of APP and elevation of its
amyloidogenic A β product in old age (Basha et al., 2005). We also observed elevations in the
oxidative DNA marker 8-hydroxy-2’-deoxyguanosine (8-oxo-dG) in older rats that had been
developmentally exposed to Pb (Bolin et al., 2006). These findings suggested that
environmental influences occurring during brain development pre-determined the expression,
regulation, and processing of APP later in life, potentially influencing the course of
amyloidogenesis and oxidative damage.
In order to link these molecular and oxidative perturbations observed in rats to pathological
consequences associated with AD, we have examined the brains of aged Cynomolgus monkeys
who were similarly exposed to Pb as infants. Primates are among a few animal models that
express amyloid plaques and other pathological features that are absent in wild-type/non-
transgenic rodents. This study was undertaken to determine whether non-human primates
which exhibit similar AD-like pathology in old age (Price and Sisodia, 1994) would be
influenced by developmental perturbations, and to explore the potential mechanisms that could
mediate such latent effects.Materials and Methods
Animal Exposure
In 1980?81, a cohort of female monkeys (Macaca fascicularis ) was randomly assigned at birth
to one of two exposure groups: one received 1.5 mg/kg/day of lead acetate (Pb) from birth until
400 days of age via infant formula and vehicle after weaning, while the other group served as
a control group and received formula or vehicle only. No overt signs of toxicity or health related
problems were evident in the animals as a result of Pb exposure (Rice, 1990, 1992). They were
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then transferred to NIH facility until termination in 2003 at approximately 23 years of age and
all animal procedures were conducted under the supervision of a licensed veterinarian
according to a NIEHS/NIH approved animal protocol. As previously reported, the blood lead
levels of these animals at 400 days of age averaged 19?26 μg/dl in Pb exposed monkeys as
compared to 3?6 μg/dl in the controls (Rice, 1990, 1992). The monkeys were terminated in
2003 (23 years later) and multiple organ tissues, including the brain were collected, cut in 1cm
sections, and immediately processed in 10% formalin for histopathology or frozen on dry ice
and stored at ?80°C. At this time, the animals were in good health and there were no indications
of adverse health effects as a result of early Pb exposure.
Total RNA isolation, synthesis of cDNA, and Real Time PCR
RNA from various control and exposed monkey cortical tissues was isolated according to the
TRIzol method (Invitrogen, CA). The RNA was reverse transcribed to obtain cDNAs,
catalyzed by SuperScript III Reverse Transcriptase (RT). The RNA/primer mixture containing
500 ng of total RNA, 1μL of 10mM dNTP mix and 1μL Oligo(dT) was incubated at 65°C for
5 min. A reaction mixture containing 2 μL of 10× RT buffer [200 mM Tris-HCl (pH 8.4), 500
mM KCl], 4 μL of 25 mM MgCl 2, 2 μL of 0.1 M DTT and 1 μL of RNaseOUT recombinant
RNase inhibitor (40 U/μL) was added. One microliter of SuperScript III RT (200 U/μL) was
then added and incubated at 50°C for 50 min. The reaction was terminated at 85°C for 5 min.
One microliter of RNase H was added and the reaction was incubated for 20 min at 37°C. The
resulting cDNA was stored at ?20°C and used in the real time PCR step. The primer pairs used
for APP, Sp1, BACE1 and GAPDH were as follows. Sp1 sense: 5’-CAA GCC CAA ACA
ATC ACC TT-3’; antisense: 5’-CAA TGG GTG TGA GAG TGG TG-3’. BACE1 sense: 5’-
TTT GTG GAG ATG GTG GAC AA-3’; antisense: 5’-CAG CAC CCA CTG CAA AGT
TA-3’. APP sense: 5’-GCT GGC TGA ACC CCA GAT-3’; antisense: 5’-CCC ACT TCC CAT
TCT GGA CAT-3’. GAPDH sense: 5’-TGA AGC AGG CGT CGG AGG G-3’; antisense: 5’-
CGA AGG TGG AAG AGT GGG TG-3’. Each real time PCR reaction mix contained 1 uL of
cDNA, 1 uL of primer mix (final concentration 200 nM), 10.5 uL of nuclease free water and
12.5 uL SYBR? GREEN PCR Master Mix (Applied Biosystems, CA). Each sample had
triplicates. Real Time PCR was conducted for all of the above genes with respective primer
pairs in a 7500 Real-Time PCR System following standard protocol. That was, 50 °C 2 min
followed by 95 °C 10 min, then 40 cycles of 95 °C 15 sec and 60 °C 1min. Real time PCR
products were checked with agarose gel to confirm that no non-specific products formed.
Results were analyzed with 7500 system software with relative quantification method using
GAPDH as endogenous control. Other housekeeping genes such as beta-actin were also
utilized.
Beta amyloid (A?) 1?40 and 1?42 assay The levels of A? were measured using human A? (1?40 and 1?42) assay kits (Immuno-
Biological Laboratories, Gunma Japan). These kits were designed as solid phase sandwich
ELISA with two kinds of highly specific antibodies. The assay conditions were followed
according to the method described by Morishima-Kawashima (2000) et al., with slight
modifications. Brain tissue was homogenized in Tris-Saline (TS) [50 mM Tris-HCl buffer, pH
7.4; 150 mM NaCl; 1 μg/mL TLCK (N-Alpha-p-tosyl-L-Lysine chloromethyl ketone); 1 μg/
mL antipain; 0.5 mM DIFP (Diisopropyl fluorophosphates); 0.5 mM PMSF; 0.1% Protease
Inhibitor cocktail] and centrifuged at 100,000 g for 20 min at 4°C. The pellet was resuspended
in 4 volumes of TS and centrifuged at 70,000 g for 20 min at 4°C. The resultant pellet was
dissolved in 500 μL of 6 M guanidine-HCl (in 50 mM Tris buffer, pH 7.6), incubated at room
temperature (RT) for 30 min and centrifuged at 70,000 g for 20 min at 4°C. The resultant
supernatant was collected and diluted by EIA buffer (supplied with the kit) to 12× to reduce
sample Guanidine-HCl concentration, and aliquots (200 μg of protein in 100 μL EIA buffer)
and assay standards were added to a 96 well plate [pre-coated with anti-human A? (35?40)
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(1A10) Mouse IgG MoAb] and incubated over night at 4°C. The wells were washed 7× with
EIA buffer. Then 100 μL of labeled antibody was added to each well containing sample or
standard and incubated at 4°C for 1 hour. The wells were washed 9× with EIA buffer followed
by the addition of 100 μL of TMB buffer, and incubated in the dark for 30 min at RT. The
reaction was stopped by adding 100 μL of 1N H 2SO 4 and the colormetric absorption was
performed at 450 nm. The levels of A? in the test samples were calculated relative to the
standard curve generated on each plate.
Immunohistochemistry
The cellular distribution of APP, SP1 and A β was examined in paraffin embedded brain tissue
of Control (Con) and developmentally, Pb-exposed (Pb-E), 23-year old Cynomolgus monkeys.
The sections were subjected to brief washes in 1× phosphate buffered saline (PBS) and 3%
hydrogen peroxide. After rinsing, the sections were incubated in PBS containing 2% Bovine
Serum Albumin (BSA) and 1% Triton X-100 blocking solution for 30 min, then incubated in
the presence of primary antibody for APP (1:200; Sigma-Aldrich, St. Louis, MO), A β (1:50;
Sigma-Aldrich), or SP1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°
C. Sections were washed with PBS and incubated along with the species-specific biotinylated
secondary antibody mouse/rabbit (1:200; Vector Labs, Burlingame, CA) for 30 min. The
sections were incubated with Streptavidin (Vector Labs) for 30 min, rinsed briefly with PBS,
and immunoreactivity was detected with the substrate, 3?3′ Diaminobenzidine-
tetrahydrochloride (DAB) (Vector Labs). Coverslips were mounted with Permanent Mounting
Medium (Vector Labs).
In all cases, negative controls were run on slides from each animal by omitting the primary
antibody incubation. No signal was evident after incubation of tissues in secondary antibody
alone.
Nuclear protein extraction
Nuclear proteins were extracted from the frontal association cortical tissue of control and Pb-
exposed animals according to the method described by Dignam (1983) et al., with slight
modifications. Tissue samples were homogenized with 1 ml PBS (phosphate buffered saline,
pH 7.4) and centrifuged at 2500 g for 10 min at room temperature. The pellets obtained were
suspended in 5 volumes of Buffer A (10 mM HEPES at pH 7.9, 1.5 mM MgCl 2, 0.5 mM DTT,
0.5 mM EDTA, and 0.2 mM PMSF) and centrifuged at 6,000 g for 2 min at 4°C. The pellets
were resuspended in 3 volumes of Buffer A and centrifuged at 6,000 g for 2 min at 4°C. The
resulting pellets were then resuspended in 5 volumes of buffer C (20 mM HEPES at pH 7.9,
1.5 mM MgCl 2, 0.5 mM DTT, 0.5 mM EDTA, 420 mM NaCl, 20% glycerol, 0.2 mM PMSF,
0.002 mg/ml aprotinin, and 0.0005 mg/ml leupeptin) and homogenized. The final suspensions
were centrifuged at 12,000 g for 10 min. The supernatants were transferred to 1.5 ml tubes,
snap frozen in an ethanol dry-ice bath, and stored at ?80° C (Basha et al., 2005).
Primary mice cortical neuronal cell culture
Since the animals we studied were 23 years old and we did not have early time points to measure
the progression of the molecular or epigenetic changes, we used a cell culture model to address
some of the mechanistic questions. We used the C57BL/6 mouse as a source of primary cortical
neurons based upon the wide use of this genetic background strain for various transgenic mice
AD models. The C57BL/6 mice (Charles River Laboratories, Wilmington, MA) fetuses were
used to generate primary cortical neuronal cultures. Animal usage was approved (Approval
#AN00?01?007) and monitored by the Institutional Animal Care and Use Committee
(IACUC) of the University of Rhode Island. Brains were excised and cortices dissected from
day 15 mouse pups from the same dam. Meninges were removed and cortical tissue incubated
for 15 min at 37°C in 5 ml Hanks' Balanced Salt Solution (HBSS) containing papain (2 mg/
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ml). Tissue was centrifuged at 125 g for 5 min and the resulting pellet was resuspended in
HBSS and cells dissociated with trituration through a fire polished Pasteur pipette and repeated
3 times. Following centrifugation, cells were plated at a constant density of 6.5 × 104/well, in
24-well poly-D-Lysine pre-coated plates. The plating medium contained 2% B27, 0.5 mM L-
Glutamine and 25 μM glutamic acid in NEUROBASAL medium (Invitrogen, CA) and
maintained at 37°C in a humidified atmosphere of 5 % CO 2. After 4 days in culture, half of
the medium was replaced with fresh medium devoid of glutamic acid and the cultures were
maintained. To determine methyltransferase activity, cells in the 24-well plates were treated
with 0.1 μM Pb at the time of medium change for 24 h. Pb was removed and cells were aged
for 7 days before nuclear extract was harvested and subjected to methyltransferase activity
assay.
8-oxo-dG determination
8-oxo-dG levels were determined by HPLC as described previously (Bolin et al., 2006).
Briefly, sample tissue was homogenized in nuclease free homogenization buffer followed by
digestion with proteinase K to remove proteins. The DNA was precipitated by 3 consecutive
organic extractions, precipitation by two volumes of ethanol (with respect to the aqueous
volume), and incubated overnight at ?20°C. The purified DNA was prepared for HPLC analysis
by nuclease digestion into deoxynucleoside components. The amount of 8-oxo-dG and 2’-
deoxyguanosine (2’-dG) was calculated by comparing the peak areas of 8-oxo-dG and 2’-dG
obtained from the enzymatic hydrolysate of the DNA sample to a calibration curve for both
compounds. Levels of 8-oxo-dG in the samples were expressed relative to the content of 2’-
dG, e.g., the molar ratio of 8-oxo-dG/2’-dG (fmol 8-oxo-dG/nmol of 2’-dG). The HPLC
method used for analysis of the samples was as follows: The mobile phase consisted of 100
mM sodium acetate, pH 5.2, with 5% methanol. Flow rate was kept at 1 mL/min using a Model
582 Solvent Delivery Module (ESA, Chelmsford, MA). DNA was analyzed using a reverse
phase YMC basic HPLC column (4.6×150 mm) with a 3 μm particle size (YMC Inc.
Wilmington, NC). 8-oxo-dG and 2’-dG were detected by a Model 5600A CoulArray Detector
(ESA) with three model 6210 four-channel electrochemical cells; potentials were set at 175,
200, and 250 V for 8-oxo-dG and at 785, 850, and 890 V for 2’-dG. Data were recorded, stored,
and analyzed with CoulArray for Windows32 Software (ESA). Data were expressed as
femtomoles of 8-oxo-dG per nanomole of 2’-dG.
DNA methyltransferase assay
DNA methyltransferase activity was determined in nuclear extracts derived from the control
and Pb-exposed frontal associated cortical tissue. Nuclear extracts were derived as described
above and the DNA methyltransferase activity was assayed following the method as described
by Takiguchi (2003) et al. Nuclear extracts containing 60 μg of protein (source of
methyltransferase) were incubated with 50 ng of deoxyinosine-deoxycytidine (poly
[dI.dC].poly[dI.dC]) double stranded DNA template (Sigma) as a substrate, 1 μM 3H-labeled
S-adenosyl methionine (SAM) (79 Ci/mmol), 100 mM Tris-HCl (pH 8.2) in a final volume of
30 μL. The reaction was initiated by the addition of SAM for 1 hour at 37° C and terminated
by chilling on ice. A 15 μL aliquot was spotted on DE81 filter paper and washed with sodium
phosphate buffer (pH 7.0) twice, once with 70% ethanol, and once with 100% ethanol. The
filter was dried and counted in a scintillation counter.
Statistical treatment
Data were analyzed using two-tailed Student's t -test and the values marked with an “*” were
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RESULTS
Pb levels
The exposure of these animals to low level of inorganic Pb from birth to 400 days resulted in blood Pb levels of 19?26 μg/dL when being dosed with Pb after weaning from infant formula.This level is slightly above the 10 μg/dL considered safe for humans by the CDC (1991), and is similar to the exposure scenario we had previously used in lifetime studies with rats (Basha et al., 2005). Sampling during young adulthood demonstrated clearance of Pb from the blood and levels were similar for both control and Pb exposed monkeys (Rice, 1992). Thus, any significant exposure was limited to the developing and adolescent period. In the aged animals,Pb levels in brain tissue were analyzed using Elemental Analyzer ICP-MS and we found that Pb levels for both exposed and control animals remained below the detection levels (<0.1 ng/g wet wt. of tissue).Latent expression of APP, A β, BACE1 and Sp1The amino acid sequence of APP of Cynomolgus monkeys is homologous (96%) to that of humans making it a good model for studies of the pathological effects of A β in the primate brain (Podlisny et al., 1991). The enzyme BACE1 plays a major role in the cleavage of APP (Singer et al., 2005) and transcription factor Sp1 (Lahiri and Robakis, 1991; Christensen et al.,2004) is a known regulator of both genes. Therefore, we compared APP, BACE1, and Sp1mRNA levels in the control and developmentally Pb-exposed aged primates. The mRNA levels of all three genes (Fig. 1) were elevated in primates exposed to Pb as infants; however, the elevation in BACE1 was not statistically significant (p=0.063). Quantification of results indicated that APP mRNA levels increased 50% (p=0.045); A β1?40 levels increased 50%(p=0.011); and A β1?42 levels increased 100% (p=0.029), in a manner similar to that previously seen in rodents (Fig. 2A, 2B). While rodents have non plaque-forming A β with 3 amino acids different from that of primates, APP of Cynomolgus monkey shares 96% sequence homology with human, and both species have same plaque-forming A β peptides, which is more clinically
relevant. Furthermore, the ratio of A β1?42 to A β1?40 increased due to developmental
exposure to Pb (Fig. 2B). Exposure of cultured mouse primary neurons to these peptides
confirmed that both of these peptides were toxic to the cells even in the soluble form and that
A β1?42 was more cytotoxic than A β1?40 (Supplementary data: Figure-1).
Alteration of AD-pathology
Immunohistochemical analysis of the frontal association cortex was undertaken to determine
if the observed molecular changes in APP expression and A β levels were accompanied by
changes in the pathological features of the brains of these animals. The brains of the aged
monkeys developmentally-exposed to Pb revealed an increase in the intracellular staining of
total A β and dense-core plaques as compared to age-matched controls (Fig. 3). Higher
magnification of intracellular A β staining revealed the accumulation of immunoreactive A β
aggregates inside neuronal cells, the budding of some of these A β species from the membrane
as well as their deposition in the extracellular space (Fig. 3). We also observed diffuse and
cored plaques and NFTs morphologically similar to those observed in human brain. The A β
plaques were found to be rich in A β1?42, the more amyloidogenic species of A β, and were
detectable using Congo red staining.
DNA methylation
The latent expression of genes observed in these animals may be mediated through epigenetic
pathways that are regulated via DNA methylation. To determine whether developmental
exposure to Pb interfered with DNA-methylation patterns, we examined the activity of DNA
methyltransferase 1 (DNMT1) in the 23-year old primate brain tissues. The activity of this
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methylating enzyme is selective for cytosine in a CpG dinucleotide, which is base-paired to a
methylated CpG sequence on the complementary strand of DNA and is directly proportional
to the abundance of methyl groups on CpG dinucleotides in the DNA (Poirier and Vlasova,
2002; Takiguchi et al., 2003). We found the activity of DNMT1 to be reduced by about 20%
in brain tissue derived from developmentally Pb-exposed primates (Fig. 4B). Exposure of
mouse primary cells from the cortex to low levels of Pb (0.1 μM) for a transient 24 hour period
followed by aging of the cells for a week produced a similar trend in DNMT1 activity (Fig.
4A).
DNA oxidation
In addition to the alteration in APP and A β deposition, age-related accumulation of oxidative
damage is suspected to play a role in the pathogenesis of AD. A β levels are well known to
induce functional disturbance in vivo through their pro-oxidant and neurotoxic properties
(Castellani et al., 2006). Along with the increase seen in A β levels in the cortex of
developmentally Pb-exposed animals, higher levels of the biomarker of oxidative DNA
damage, 8-oxo-dG were noted (Fig. 5).Discussion The data presented in this paper show that monkeys exposed to Pb at birth to 400 days up-regulate the expression of APP, BACE1, and Sp1 in old age. The up-regulation of both APP and BACE1 gene expression is mediated by Sp1, and the essentiality of Sp1 as a mediator of these delayed transcriptional up-regulations has been previously shown by us (Basha et al.,2005). Moreover, both APP and BACE1 are rich in CpG dinucleotides and GC box elements (Pollwein et al., 1992) making them subjected to epigenetic reprogramming and transcriptional regulation via DNA methylation pathways (Lahiri et al., 2007). This is further reinforced by preliminary microarray screens which showed that more than 95% of the differentially expressed genes screened in control versus Pb-exposed monkeys were also CpG-rich
(supplementary data: Table-1). This transcriptional reprogramming at the gene level is also
translated into biological consequences. Levels of amyloidogenic products of APP were also
increased in the aged animals that were exposed to Pb as infants (Fig. 2A). This is consistent
with earlier findings in rodents (Basha et al., 2005) and argues for a transcriptional process that
promotes neurodegeneration in old age.
These molecular and biochemical changes observed in 23-year old animals are accompanied
by altered features of AD-like pathology in the exposed monkeys. Our intracellular staining
closely resembles what is seen in humans and other animal models (Mochizuki et al., 1996;
Schmitz et al., 2004). It reveals granular, oval, and crescent shaped A β localization in pyramidal
cells and globular shaped neurons in layers II-IV of the cortex. The occurrence of these
molecular, biochemical and pathological changes in primates that develop plaques and tangles
in old age, in response to developmental exposure to Pb, suggests that developmental exposure
can influence latent pathogenesis, hence bearing a direct relevance to humans. The possibility
that developmental exposure to Pb could result in the formation of AD pathology in humans
is further supported by findings in a patient who survived from severe Pb toxicity at 2 years of
age, but died of severe mental deterioration at the age of 42 (Niklowitz and Mandybur,
1975). The brain of this patient revealed many senile plaques and NFTs (Niklowitz and
Mandybur, 1975). Although this is a single case, the plausibility that early exposure to Pb could
be a risk factor for AD warrants further study.
One way to achieve permanent changes or long-term alteration in gene expression is to alter
the structural make-up of the DNA bases that determine the sequence-specific DNA-binding
of a transcription factor. Our findings with changed DNA methyltransferase activity argue that
exposure to Pb early in life interferes with gene imprinting and could thus leave a permanent
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molecular scar on the DNA. Consistent with such a potential mechanism is the decreased activity of the DNA methylating enzyme in the aged animals exposed to Pb as infants, as well as the delayed decrease in the activity of this enzyme in cells that had prior exposure to Pb (Fig. 4).This is further supported by studies that show that regions of the human APP promoter upstream of ?500bp displayed tissue and brain region-specific profiles of methylation, which roughly reflect APP expression patterns (Rogaev et al., 1994) and age-related reduction in methylcytosine (?224 to ?101) that occur on the human APP promoter (Tohgi et al., 1999).To link the association of an epigenetic phenomenon in current model, we hypothesize that genes that are regulated by methylation can be reprogrammed in adulthood due to infantile exposure to inorganic Pb. This hypothesis was supported by our microarray screening of 588neurobiology-related genes. We found that most of the genes (20 out of 22) that were altered due to infantile exposure to Pb were rich (>60%) in CpG dinucleotides (Supplementary data:Table-1). However, our own work herein and elsewhere (Basha et al., 2005) has shown that while SP1 and APP are up-regulated in a latent fashion after early exposure to Pb in both Cynomolgus monkeys and rats, other genes regulated by SP1, such as BACE1 (Christensen et al., 2004) do not respond in rodents (Bolin et al., 2006) and show a modest trend in primates.In addition, SP1 regulates a wide variety of other genes that have not been shown to respond in this latent fashion to early Pb exposure. This does not exclude SP1 from an active role in latent Pb-induced pathogenesis. The specific position of an affected SP1 site may explain the difference. For example, active SP1 sites in the APP gene appear in its 5’-UTR (Villa et al.,2004) while the active SP1 site in the BACE1 promoter is approximately 1kb upstream of the transcription start (Christensen et al., 2004). Alternatively, general density of CpG dinucleotides in combination with presence of transcription factor sites (such as SP1) may explain the differential effects between APP and BACE1. Comparison of the cumulative potential methyl CpG dinucleotides 2 kilobases (kb) upstream of the transcription start sites
indicated that the proximal promoter regions surveyed by Bolin et al suggested a difference in
methylation site density centered around ?100 (Bolin et al., 2006).
Ample evidence has accumulated that oxidative damage to macromolecules such as DNA,
protein, and lipids (Cecchi et al., 2002; Esposito et al., 2006), as well as, a down-regulation in
antioxidant enzymes are associated with AD (Smith and Perry, 1995). We have previously
found elevations in the oxidative DNA marker 8-oxo-dG in older rats that had been
developmentally exposed to Pb (Bolin et al., 2006). Here we also find a similar accumulation
of 8-oxo-dG (Fig. 5). This latent accumulation of oxidized DNA could possibly come from
two sources. The latent increase in A β could promote the formation of reactive oxygen species,
thus damaging the DNA; and/or epigenetic modulation in the methylation pattern of cytosines
could interfere with the repair of oxidized guanines or render them more susceptible to
oxidative damage (Evans and Cooke, 2004).
Few studies address both DNA methylation and DNA oxidative damage as an epigenetic
phenomenon. Researchers using synthetic DNA oligonucleotides with both methylation and
oxidative damage in a single CpG site or oxidized dG at a complementary strand have found
oxidation of guanine in a CpG dinucleotide reduced the MBD (methyl group binding domain)
binding to that site (Valinluck et al., 2004). Even the oxidation on the guanine in the opposite
strand diminished the MBD binding but not so much as the same strand G-oxidation. When 5-
methylcytosine was oxidized to 5-hydroxymethylcytosine, its affinity to MBD is greatly
reduced to the same level as unmethylated cytosine. We thus hypothesize that epigenetic
mechanisms such as DNA methylation can influence oxidative damage and make organisms
more susceptible to the pathogenesis of AD and that this process can be modulated by
environmental exposure.
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We, therefore, propose a model (Fig. 6) of interaction between methylation and oxidative
damage in genes reprogramming. Using the APP gene proximal promoter/5’-UTR sequence
as an example, in undamaged genes, MECP2 (methyl CpG binding protein 2) would compete
against transcription factors such as SP1 for regulation. The resulting competition would down-
regulate a given gene. If target DNA is altered via oxidation to 8-oxo-dG or 5-
hydroxymethylcytosine, MECP2 binding may be partially or completely blocked, but
insufficiently to permit sufficient competing SP1 interaction with the sequence to change
expression (Fig. 6B). Normal changes of development, maturity, and aging result in triggering
of latent SP1 regulatory alterations, which significantly increase levels of the transcription
factor (Fig. 6C). The greater levels of SP1, when combined with partial blockage of MECP2
binding, permit sufficient SP1 interaction with the sequence to result in greater target gene
expression. It is likely that other transcription factors may function as competitive targets for
MECP2 in addition to SP1.
Adult diseases such as schizophrenia have also been linked to infection, fetal malnutrition or
hypoxia in early life (Dalman et al., 1999; Van Erp et al., 2002; Boksa and El-Khodor, 2003).
A study by Bilbo et al . (2005) showed that perinatal exposure to an infectious agent affected
how the nervous system responded to an immune challenge and memory consolidation later
in adulthood. The authors also found that neonatal pathogen exposure decreased the number
of adult hippocampal astrocytes, increased their reactivity, and decreased brain IL-1β levels
following adult lipopolysaccharide (LPS) exposure. Work in autism spectrum disorders has
demonstrated at least some contribution of differential methylation (Jiang et al., 2004).
Hypomethylation of the DRD2 and HTR2A genes have been implicated in both schizophrenia
and bipolar disorder (Abdolmaleky et al., 2004). On the other hand, hypermethylation of the
RELN gene has been shown to associate with schizophrenia (Abdolmaleky et al., 2005),
indicating that latent pathogenic effects of epigenetic perturbations may work on more than
one mechanism, while our work specifically addressed latent effects of Pb, other metals may
have a similar effect (Poirier and Vlasova, 2002 ;Takiguchi et al., 2003).
In conclusion, this paper presents novel findings in primates that implicate an environmental
agent (Pb) in the pathogenesis of AD and demonstrate that development is an important period
of vulnerability which could increase future susceptibility to neurodegeneration and AD
pathology.
Acknowledgements
This research was supported by grants (ES013022 and AG027246) from the National Institutes of Health (NIH)
awarded to NHZ and URI core facility grant (P20RR016457) funded by the National Center for Research Resources
(NCRR), a component of NIH. Work at FCP's lab was supported by the division of intramural research of NIEHS/
NIH, NIA grant1R15AG023604-01; and work at DKL's lab was supported by NIH P20 RR15583-07 and NIH
P20RRP20RR017670-04, Alzheimer's association and NIH grant AG18379 and AG18884.References
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Supplementary Material
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Figure 1. Changes in mRNA expression of APP, BACE1 and Sp1 in the frontal association cortex
of aged monkeys following developmental exposure to Pb
The frontal association cortical tissue of 23-year old Control (Con) and Pb-exposed (Pb-E)monkeys were analyzed for mRNA expression of APP, BACE1, and Sp1. The mRNA
expression of APP, BACE1, and Sp1 were validated by real time PCR. Data shown represent the mean ± S.E.M. (4 animals in each group). Values marked with an “*” are significantly different from their corresponding controls (p <0.05) as determined by student's t-test.
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Figure 2. Elevation of A β levels in the frontal association cortex of 23-year old Cynomolgus monkeys
following developmental exposure to Pb
Brain tissue of Control (Con) and Pb-exposed (Pb-E) 23-year old Cynomolgus monkeys were used for the analysis of A β levels (A-B). The levels of A β were measured in the frontal association cortical tissue using ELISA as described in the methods section. Data shown represent the mean ± S.E.M. for 4 animals in each group. Values marked with an “*” are
significantly different from their corresponding controls (p <0.05) as determined by a student's t- test.
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Figure 3. Photomicrographs showing AD-like pathology in the frontal association cortex of 23-year
old Cynomolgus monkeys following developmental exposure to Pb
Brain tissue of Control (Con) and Pb-exposed (Pb-E) 23-year old Cynomolgus monkeys were used for the analysis of immunohistochemical analysis of AD-like pathology. Sections were prepared and stained with the A β-specific antibody which recognizes both A β1?40 and A β1?42 as discussed in the methods section. Arrows point to A β-containing plaques as well as granular and intracellular staining. The staining characteristics are similar to those reported in the literature.
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Figure 4. DNA methyltransferase activity in cortical neuronal cells of mice and in monkey brains
(A) Control and Pb-exposed mouse cortical neuronal cells, and (B) Frontal association cortical tissue of 23-year old control (Con) and Pb-exposed (Pb-E) monkeys were used to estimate the DNA methyltransferase activity as described in the methods section. Data shown represent the mean ± S.E.M. for 4 independent determinations (4 animals in each group for monkey data).Values marked with an “*” are significantly different from their corresponding controls (p <0.05) as determined by a student's t -test.
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Figure 5. Oxidative DNA damage in control and infantile-exposed aged monkey brains Frontal association cortical tissues were obtained from 23-year old control (Con) and Pb-exposed (Pb-E) monkeys and used to measure the marker of oxidative DNA damage as described in the methods section. Data shown represent the mean ± S.E.M. for 4 animals in each group. Values marked with an “*” are significantly different from their corresponding controls (p <0.05) as determined by a student's t - test.
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Figure 6. Model of effects of oxidative damage on SP1-mediated expression of APP via interference with MECP2 binding of methylated CpG dinucleotides The APP promoter and 5'-UTR from ?1000 base pairs to the “ATG” start codon at +148 are shown. CpG dinucleotides are indicated on the sequence, as is the +1 transcription start site.(A) Undamaged DNA, showing the binding of one or more MECP2 to methylated CpG,competing against SP1. APP expression is at normal levels. (B) Conversion of G residues to 8-oxo-dG or mC to 5-hydroxymethylcytosine blocks binding of some MECP2 to methylated CpG, potentially permitting additional binding of SP1 but with insufficient levels to significantly alter target gene expression levels. (C) Normal changes of development, maturity and aging result in triggering of latent SP1 regulatory alterations, which significantly increase
levels of the transcription factor, permitting SP1 to out-compete remaining MeCP2 binding,increasing expression of target genes.
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常用工具安全操作规程(一)
常用工具安全操作规程(一) 1、常用工具主要指：台钻、手电钻、砂轮、扳手、手锤、锉刀、手锯、凿子、冲子、螺丝刀、虎钳、千斤顶、刮刀等。 2、凡使用上述工具都必须熟知并认真执行本规程。 一、台钻 1、工作前应扎紧袖口，戴好帽子，女工必须将头发塞进帽子里。操作时严禁戴手套及围巾，手中不准拿回丝、花包布等物。 2、工作前先检查机器、工具、电气设备和安全防护装置是否完好。 3、工作时精力要集中，严禁手触动正在转动的任何部位。变换车速、测量工件以及装卸工件、钻头、夹具等都必须在机器停稳后进行。 4、工件必须垫稳、夹牢。必要时要在工件下面垫垫块。 5、将要钻透工件时，应减慢进刀速度。 6、钻孔时工作台面不准放置刀具、量具及其他物品。 7、松紧钻头必须用钻头钥匙，不准用锤子或其他东西敲打。 二、手电钻 1、认真执行台钻有关安全操作规程。 2、手电钻应使用三芯（单相）、四芯（三相）橡胶软线，出线头必须有胶圈固定，电线长度不得超过10米。 3、使用前必须认真检查，发现电线绝缘不良，插头破裂、无接地线等情况禁止使用。接电源必须先查清电源电压与电钻电压是否相符。 4、使用手电钻要戴绝缘手套或站在绝缘台上操作。工作时如发现漏电、
震动、异响或温度过高等现象，必须立即停止使用，交电工检修。5、使用手电钻时要保持身体平衡，持稳电钻，不可晃动，工作时用力要适当。 6、工作中，不准把电线缠在肩上、臂上。移动手电钻时，必须手握钻柄，严禁拖提电线。 7、手电钻所用的导线，必须保证不受其他物体撞击，碾压和割戳，在车辆通过的地方，电线必须架空。 8、在高处作业使用手电钻必须有安全措施，在机器内或金属容器内使用手电钻必须有人监护。 三、砂轮 1、使用砂轮必须戴防护眼镜，使用前应先检查砂轮有无裂纹，安全罩是否牢固，夹板和螺丝是否松动，有缺陷不得使用。 2、工作前应先试转，开动砂轮转到正常速度，观察砂轮无异常情况时，才准许磨工件。 3、磨工件必须用双手把工件握牢，不得用力过猛或使工件在砂轮上跳动。 4、磨笨重或较大的工件，必须有支架，以免撞击。严禁用砂轮侧面磨工件。 5、砂轮和防护罩之间，不得落下杂物，如落下杂物必须立即关车处理。 6、操作者必须站在砂轮侧面，把头闪开，禁止两人同在一个砂轮上工作。
常用修保机具(工具)的使用安全操作规程示范文本
常用修保机具（工具）的使用安全操作规程示范文 本 In The Actual Work Production Management, In Order To Ensure The Smooth Progress Of The Process, And Consider The Relationship Between Each Link, The Specific Requirements Of Each Link To Achieve Risk Control And Planning 某某管理中心 XX年XX月
常用修保机具（工具）的使用安全操作 规程示范文本 使用指引：此操作规程资料应用在实际工作生产管理中为了保障过程顺利推进，同时考虑各个环节之间的关系，每个环节实现的具体要求而进行的风险控制与规划，并将危害降低到最小，文档经过下载可进行自定义修改，请根据实际需求进行调整与使用。 1、手锤和大锤 （1）打锤前，应检查锤头与锤把安装是否牢固，手 柄有无裂纹、油污，以防大锤滑脱伤人。开裂或起毛刺的 锤头不得使用。 （2）使用大锤时必须注意安全，四周要有足够的空 间，后方和正前方不得有人，严禁戴手套打锤。 2、电气设备及移动式电动工具 （1）性能良好，防护设施齐全有效，有专人负责 （保管）。定期检查电气设备（工具）绝缘情况，如有漏 电现象，应停止使用，并立即进行检修。 （2）有与电气设备（工具）相配专用闸刀、插头、插
座、漏电保护器等，且完好有效。 （3）设备（工具）至电源的的导线，应采用橡套电缆，单相设备用三芯线，三相设备用四芯线。其中一芯做为接地导线，两端分别接牢设备壳体和总接地极。 （4）定期检查橡套电缆线老化程度，若裂纹、破损应立即更换，以免漏电。 （5）操作移动电动工具时应戴绝缘手套穿绝缘鞋。 3、液压千斤顶安全使用 （1）工作稳定性能良好，无漏油、泄油现象。 （2）在使用液压千斤顶时，基础必须稳固可靠，载荷应与千斤顶轴线一致。在作业过程中，严防千斤顶偏斜造成被承物倒塌。 （3）液压千斤顶的顶头与汽车（设备）的金属面接触时，应垫硬木块，防止滑脱。 （4）液压千斤顶的顶升高度不得超过有效顶程。
共同但有区别责任原则
题目：共同但有区别的责任原则在实施中的困境与对策姓名：罗珠玉、戴政
共同但有区别的责任原则在实施中的困境与对策 摘要：共同但有区别的责任原则作为国际环境法的一项基本原则，该原则的要求在实践中未能得到充分尊重与落实。笔者通过对该原则实施困境及原因的分析,寻求解决该原则的可行性办法。 关键词：共同但有区别的责任原则；实施困境；可行性办法
共同责任和区别责任组成了共同但有区别责任原则。二者之间相辅相成，密不可分。一方面各国不能以任何的借口而拒绝参与环境保护问题，这是每个国家的共同责任；另一方面，基于合理性而产生的区别责任，我们在对待共同责任的同时要给予发达国家与发展中国家差别待遇。只有当我们正确的理解二者关系时才能确保该原则的正确实施。实践中，该原则面对来自不同国家的阻力。 一、共同但有区别的责任原则的实施困境 发达国家有先进的技术与雄厚的资金，在各国订立国际公约之初，对发达国家明确规定了需向发展中国家提供环保技术的援助。可公约本身并未说明具体的援助方式，使发达国家有机可趁，利用市场操作以高价的方式向发展中国家提供商业性援助。而即使存在无偿性援助，实际数据也令人心寒，发展中国家适应气候变化每年所需的资金大约在 500 亿美元，而联合国的专门基金从发达国家筹集到的资金从 90 年代初至今总计只有 670 亿美元，发达国家对发展中国家的资金援助可见一斑，这也是共同但有区别责任难以落实的一个重要原因。 在发展中国家共同但有区别的责任原则的实施也受到了挑战。发展中国家的经济水平比较落后，他们没有先进的技术支撑他们在保证解决自己温饱问题的同时兼顾环境保护，而要想解决生存问题必须以牺牲坏境为代价。传统的经济发展技术、能源技术已经不能适应现代可持续发展的要求，尤其 21 世纪对各国高新技术提出了更高的要求，在环境治理方面也不例外。现在单纯的现有技术转让已经不能满足发展中国家环境治理的需要了，发达国家需要尽可能地多与发展中国家进行技术交流与合作，让发展中国家也成为高新技术开发的参与者，掌握自主的知识产权。 最后，为应对国际环境的问题而制定的众多国际公约，足以应对坚持和实施共同但有区别的责任原则。比如《人类环境宣言》、《联合国气候变化框架公约》、《联合国海洋法公约》、《京都议定书》······这些制定与签署的国际公约，不仅构成了世界环境保护国家合作的标准，而且也未共同但有区别责任作出了各种细化的规定。 二、共同但有区别的责任原则实施中存在困难的原因 美国曾以不符合本国的国家利益为由退出《京都议定书》，而各国对其只能进行谴责，因为国家享有主权原则，有权决定自己是否愿意加入某一国际公约。
钳工常用工具使用操作规程
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三、行灯： 1、使用行灯，必须绝缘良好，用前要进行检查，以防漏电。 2、使用行灯必须爱护，以免损坏绝缘，发现绝缘损坏时，应立即处理或更换。 3、行类电源电压不得超过36V，用时要做好接地保护。 四、手电钻： 1、使用手电钻时，必须戴好绝缘手套，找电工接线，严禁私自乱接。，在潮湿的地方工作时，必须站在橡皮垫或干木板上。 2、电钻外壳必须有接地线或接中性保护，电钻导线要保护好，禁止乱拖乱拽或拖在油水中，以防割破或腐蚀电线。 3、使用当中如发现漏电、震动、高热或异声时，应立即停止工作，找电工检查修理。 4、电钻未完全停止转动时，不能卸、换钻头，停电休息或离开工作地时，应立即切断电源。 5、如用力压电钻时，必须使电钻垂直工件，而且固定端要特别牢固。 五、油压千斤顶： 1、油压千斤顶必须爱护使用，做好日常维护保养工作，及时加油。 2、使用时，下面必须加平垫木，受力点要选择适当，
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论共同但有区别责任原则
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常用通用工具安全技术操作规程
常用通用工具安全技术操作 规程 -标准化文件发布号：（9456-EUATWK-MWUB-WUNN-INNUL-DDQTY-KII
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论国际环境法的共同但有区别责任原则
目录 毕业论文诚信承诺书 (2) 摘要 (3) 关键字 (3) 正文 一、共同但有区别责任原则的概述 (3) 二、共同但有区别责任原则的发展 (4) 三、共同但有区别责任原则的性质 (5) 四、共同但有区别责任原则的意义 (6) 五、坚持和发展“共同但有区别责任”原则 (6) 结语 (7) 参考文献 (7)
毕业论文诚信承诺书 本人作为《论国际环境法的共同但有区别责任》一文的作者，郑重承诺： 一、本论文是我在导师的指导下，参考相关文献资料，进行分析研究，独立完成的，其中所引用的文献资料和相关数据，都是真实的，除标明出处的内容外，不包含他人已公开发表的研究成果和学术观点。 二、本论文中若有抄袭他人研究成果和剽窃他人学术观点，本人自愿承担取消毕业论文成绩、交回学历学位证书等一切后果。 学生签名： 年月日注：本承诺书一式二份，一份置于毕业论文分册首页，一份置于过程材料分册末页。
论国际环境法的共同但有区别责任原则 摘要 环境保护已经成为我们时代最为重大的主题之一。世界每一个成员都应当共同承担保护和改善全球环境的责任，环境保护不再是仅限于一个两个国家主权之间的事情。全球性环境问题需要所有国家的共同努力才能得以解决。在对环境问题形成所起的作用上，发达国家和发展中国家扮演者主次不同的角色。如果要让本来就相对贫困的发展中国家在解决目前的全球性问题上承担和发达国家同样的义务，肯定是不公平的，必然会遭到发展中国家的反对。国际环境法的共同但有区别的责任原则“就在这样的背景下产生了，它调解了国家之间的矛盾，促进各国都参与到全球环保事业当中，将不同国情，制度的国家团结成一个“求大同、存小异”合作的整体。共同担有区别的责任原则主要包含两层意思：共同责任和区别责任。它不但是国际法上的一项重要原则，更代表了环境争议思想在适用范围上的扩展，本文主要探讨共同但是有区别的责任的定义、内涵以及环境正义与共同但有区别的责任之间的关系，还有共同但有区别的责任定义的必要性。 关键字 共同但有区别的责任国际发达国家发展中国家 一、共同但有区别责任原则的概述 国际环境法中共同但有区别责任原则因体现谋求优先发展经济的利益诉求,获得大部分发展中国家认同。但该原则的地位、内容一直存有争议。随着中国、印度等发展中大国碳排放日益增长,在后续气候谈判中如仅以发展中国家身份不参加实质减排,将面临极大压力。因此,探讨该原则的地位以及承担责任的依据,对于确定发达国家与发展中国家应对气候变化所应承担的共同责任以及各自应承担的义务,将具有重要意义. 共同但有区别责任主要体现在国际气候大会中表现最为明显，备受关注的联合国第十九次气候变化大会雨2013年11月23日晚在波兰首都华沙落幕，会期比原计划拖延了一整天。经过长达两周的艰难谈判和激烈争吵，特别是会议结束前最后48小时，各国代表挑灯夜战，最终就德班平台决议、气候资金和损失损害补偿机制等焦点议题签署了协议。 但是，由于发达国家不愿承担历史责任，在落实向发展中国家提供资金援助问题上没有诚信，导致政治互信缺失，加上个别发达国家的减排立场严重倒退，致使谈判数次陷入僵局。会议最终经过妥协，达成了各方都不满意、但都能够接受的结果。 《联合国气候变化框架公约》第十九次缔约方会议暨《京都议定书》第九次缔约方会议23日晚打破僵局达成协议后在华沙落下帷幕。尽管大会成果不尽如人意，但中方表示，节能减排是中国可持续发展的内在要求，无论谈判进展如何
汽车修理工常用工具安全操作规程通用版
操作规程编号：YTO-FS-PD645 汽车修理工常用工具安全操作规程通 用版 In Order T o Standardize The Management Of Daily Behavior, The Activities And T asks Are Controlled By The Determined Terms, So As T o Achieve The Effect Of Safe Production And Reduce Hidden Dangers. 标准/ 权威/ 规范/ 实用 Authoritative And Practical Standards
汽车修理工常用工具安全操作规程 通用版 使用提示：本操作规程文件可用于工作中为规范日常行为与作业运行过程的管理，通过对确定的条款对活动和任务实施控制,使活动和任务在受控状态，从而达到安全生产和减少隐患的效果。文件下载后可定制修改，请根据实际需要进行调整和使用。 1、钳工台（桌） 1）、钳工台（桌）一般紧靠墙壁，人站在一面工作，对面不准有人。如大型钳台对边有人工作时，钳台上必须设置密度适当的安全网，钳台（桌）必须安装牢固，不得作铁铮。 2）钳台（桌）上使用照明灯电压不得超过36伏。 3）钳台（桌）的上杂物要及时清理，工具和工件要放在指定地点。 2、台虎钳 1）台虎钳上不要放置工具，以防滑下伤人。 2）使用转座台虎钳工作时，必须把固定螺丝扳紧。 3）台壶钳的丝杆、螺母要经常擦洗和加油，并保持经常清洁。如有损坏，不得使用。 4）钳口要保持完好，磨平时要及时修复以防工件滑脱。钳口紧固螺丝要经常检查，以防松动。不准使用已滑
“共同但有区别的责任”原则的解读
龙源期刊网 https://www.wendangku.net/doc/9117945562.html, “共同但有区别的责任”原则的解读 作者：王小钢 来源：《中国人口·资源与环境》2010年第07期 摘要哥本哈根气候变化会议中最大的立场之争可能是关于“共同但有区别的责任”原则的政治辩论。“给不平等者以不平等”和“给平等者以平等”是“共同但有区别的责任”原则的哲学基础。历史责任、矫正正义和“与能力有关的责任”体现了“给不平等者以不平等”的理念。人均排放权和平等参与权则体现了“给平等者以平等”的理念。在“共同但有区别的责任”原则视域中，不是中国，而是丹麦和美国劫持了哥本哈根气候变化会议。从中国的立场看，国际社会在哥本哈根气候变化会议之后理应在“给不平等者以不平等”和“给平等者以平等”理念基础上坚守“共 同但有区别的责任”原则。首先，国际社会应将历史累积排放量和人均GDP作为适应气候变化的参考标准。其次，鉴于发展中国家的发展律令和后代人的正当需要，国际社会应将人均累积排放量和人均排放量作为减缓气候变化的参考标准。最后，国际社会必须按照平等参与原则开展将来的国际谈判。 关键词气候变化；共同但有区别的责任；给不平等者以不平等；历史责任：人均标准 中图分类号X22 文献标识码 A 文章编号1002－2104(2010)07－0031－07 doi：10.3969／j.issn.1002-2104.2010.07.005 2009年12月19日，哥本哈根气候变化会议落下帷幕。《联合国气候变化框架公约》(以下简称《公约》)各缔约方均不太满意，尽管缔约方会议同意“注意到”(taking note of)《哥本哈根协议》(Copenhagen Accord)。由于苏丹、委内瑞拉和玻利维亚等国家的反对，缔约方会议没有通过《哥本哈根协议》。在联合国条约中，“注意到”的术语意味着缔约方会议没有批准也没有通过，不持肯定态度也不持否定态度。哥本哈根会议中最大的立场之争可能是关于“共同但有区别的责任”(Common But Differentiated Responsibilities)原则的政治辩论。中国、印度、巴西和南非(BASIC四国)在多次谈判场合重申坚持“共同但有区别的责任”(Common But Differentiated Responsibilities)原则。然而，美国总统奥巴马(Barack Obama)甚至在12月18日领导人会议上发言将“共同但有区别的责任”修改为“共同但有区别的回应”(Common But Differentiated Responses)。在气候正义的视角下，全球气候体制中“共同但有区别的责任”原则的哲学基础究竟是什么?在“共同但有区别的责任”原则视域中，究竟是哪些国家劫持了哥本哈
安全操作规程77567知识讲解
安全操作规程77567
xxx纺织有限公司 安全操作规程 职工安全守则 1.严格执行安全制度和安全操作规程，不违章作业，并随时制止他人违章作业。 2.上班前不准喝酒。不准带孩子进工作场所。不准将自行车推进车间。 3.女职工不留过肩的长发，进车间必须将头发塞进帽子内。 4.工作时必须按规定穿好防护用品。 5.工作时不准穿高跟鞋和赤脚、赤膊；不准围围巾、穿裙子；不准披散衣服，要扣齐纽扣，扎好袖口，把衬衣塞进裤腰里。 6.工作要思想集中，坚守岗位，不准闲谈和开玩笑。在车间里严禁打闹，追跑、打盹、睡觉。 7.不准操作和触动自己不熟悉的机器设备和工具仪器。未经领导同意不准私自调换工种和代替别人操作。 8.要爱护和正确使用安全装置及劳动保护设施，不准拆除和不用；发现损坏，立即报告修理。 9.发现机器有异响或在车间里闻到烟火味，应立即报告领导或通告有关人员检查。 10.严禁在禁烟区吸烟。在厂区动用明火必须经保卫部门同意，并严加监护。 11.不准私自取用汽油或其他危险物品。
12.原料成品库、锅炉房、变电所、配电室、危险物品库、风机室等要害部门，非本部门工作人员，不准擅自进入。 13.一切电气设备的拆装、检查、修理都必须由电工进行，非电气工作人员，不准乱动。 14.不准在电机、电线上拉铁丝、绳子和烘烤、晾晒衣物，不准在开关箱内放任何物件。 15.不准在车间内、走廊和公共场所的地面上吐痰、泼水、泼菜汤和乱丢瓜果皮核；油脂撒在地上要随时擦干净。 16.工作地点要整洁，通道不准堵塞，原材料、成品、半成品、工具、容器、车辆、机件等要放在指定地点，摆放整齐和稳妥、牢固。废料要及时清理。 17.出入车间和通道的门要了望慢行，注意安全信号和来往车辆、行人。 18.变电所附近、电线下面和厂区通道上，禁止打球、踢球。 19.非有关工作人员，不准上房顶，不准进入基建工地。 20.不要站在容易滚动、倾倒、断裂或塌陷的物件上工作。
论共同但有差别责任原则
黑龙江大学自学考试法律专业本科 毕业论文 题目：论共同但有差别责任原则作者：吴春刚 所在单位： 指导教师：李艳岩 黑龙江大学 2008年10月18日
目录 内容摘要 (1) 一、共同但有差别责任的体现 (1) 二、共同但有差别责任原则的内容 (2) 三、共同但有区别的责任原则的合理性分析 (2) 四、共同但有区别责任原则的正义基础 (3) （一）正义的一般含义 (3) （二）环境正义观念的起源 (4) （三）承担责任的实际能力分析 (4) 五、共同但有区别的责任原则在实践中的贯彻状况 (5) 六、落实共同但有差别责任原则的努力 (6) 七、结束语 (7) 参考文献 (9)
论共同但有差别责任原则 摘要：国际环境法是一个正在迅速发展的法学部门，国际环境法基本原则的体系也正在形成之中，共同但有差别责任原则的涵义分为共同的责任和有差别的责任，基于环境与人类的密切关系，在环境领域，全球所有国家对地球环境污染负有共同的防治责任，但是，由于历史的原因，发达国家只顾发展，不顾环境，大量使用环境资源和大量排放废弃物造成全球环境恶化，另发达国家在以全球环境资源的大量损耗和环境状况的急剧恶化为代价积累起来丰富的技术和财力资源，基于环境法的“谁破坏，谁治理”的公平原则，发达国家理应承担比发展中国家更为主要的或承担更多的义务。此原则在一系列国际环境法规中得到了体现。 关键词：国际环境法共同责任区别责任环境 一、共同但有差别责任的体现 保护和改善全球环境昰国际社会所要承担的共同义务。作为国际社会主要成员的各个国家，自然负有保护和改善环境的共同责任。1967年的《外层空间条约》和1971年的《禁止在海洋床底及其底土安置核武器和其他大规模毁灭性武器条约》都承认了人类对环境的“共同利益”，要实现共同利益必然要求各国承担共同责任。[1]1972年斯德哥尔摩《人类环境宣言》指出：“……保护和改善人类环境是关系到全世界各国人民的幸福和经济发展的重要问题，也是全世界各国人民的迫切希望和各国政府的责任。”关于“区别责任”，《人类环境宣言》指出：“在发展中国家中，环境问题大半是由于发展不足造成的。……因此，发展中国家必须致力于发展工作，牢记他们优先任务和保护及改善环境的必要”。1982年《内罗毕宣言》指出：“……发达国家及有能力这样做的国家，应协助受到环境失调影响的发展中国家，帮助他们处理最严重的环境问题。”《气候变化框架公约》的原则部分规定：“各缔约国应当在公平的基础上，并
常用工具安全操作规程通用范本
内部编号：AN-QP-HT784 版本/ 修改状态：01 / 00 The Procedures Or Steps Formulated T o Ensure The Safe And Effective Operation Of Daily Production, Which Must Be Followed By Relevant Personnel When Operating Equipment Or Handling Business, Are Usually Systematic Documents, Which Are The Operation Specifications Of Operators. 编辑：__________________ 审核：__________________ 单位：__________________ 常用工具安全操作规程通用范本
常用工具安全操作规程通用范本 使用指引：本操作规程文件可用于保证本部门的日常生产、工作能够安全、稳定、有效运转而制定的，相关人员在操作设备或办理业务时必须遵循的程序或步骤，通常为系统性的文件，是操作人员的操作规范。资料下载后可以进行自定义修改，可按照所需进行删减和使用。 1、常用工具主要指：台钻、手电钻、砂轮、扳手、手锤、锉刀、手锯、凿子、冲子、螺丝刀、虎钳、千斤顶、刮刀等。 2、凡使用上述工具都必须熟知并认真执行本规程。 一、台钻 1、工作前应扎紧袖口，戴好帽子，女工必须将头发塞进帽子里。操作时严禁戴手套及围巾，手中不准拿回丝、花包布等物。 2、工作前先检查机器、工具、电气设备和安全防护装置是否完好。 3、工作时精力要集中，严禁手触动正在转
试论国际环境法的共同但有区别责任原则
试论国际环境法的共同但有区别责任原则 试论国际环境法的共同但有区别责任原则 的国际责任。而另一方面发展中国家相比而言，发展起步较晚，工业化进程对全球环境的影响小，加之很多发展中国家还没有摆脱贫困的威胁，从某种意义上说发展是其的第一要义。同时发展中国家治理环境的资金技术匮乏，客观上也不具备彻底清除全球污染的实力。因此，区别责任强调，应对全球环境问题，发达国家和发展中国家的责任是有区别的，发达国家理应承担更多的、更重的责任。这也是“受益者补偿”原则的体现，同时也是维护实质正义的要求。 二、共同但有区别责任原则确立及发展 1.共同但有区别责任原则的确立。1972年在斯德哥尔摩召开的第一届人类环境大会，是国际社会就环境问题召开的第一次会议。在此次会议上通过的人类环境宣言，其内容强调和突出了利益和责任的共同性，以及具体环境和实际情况的区别，是共同但有区别责任的萌芽；1992年在巴西的里约热内卢召开的联合国环境与发展大会，是应对全球气候变化的一次会议。大会签署了《联合国气候变化框架公约》，会议提出“鉴于导致全球环境退化的各种不同因素，各国负有共同但有区别的责任”，这就正式提出了共同但有区别的责任原则，标志着共同但有区别原则的确立。根据此原则，发达国家应采取措施限制温室气体排放，同时要向发展中国家提供新的额外资金以支付发展中国家履行《公约》所需增加的费用，并采取一切可行的措施促进和方便有关技术转让的进行。 2.共同但有区别责任原则的发展。由于《联合国气候变化框架公约》没有对个别缔约方规定具体需承担的义务，也未规定实施机制，缺少法律上的约束力。因此，五年后以议定书的附属形式设定了强制排放的限制。1997年在日本京都，联合国气候变化框架公约的缔约方大会举行会议并通过了《京都议定书》，核心内容是：要求全球38个工业化程度较高的国家削减温室气体的排放量，规定了具体的减排义务。可见《京都议定书》遵循了公约确立的原则，规定了全球各国的二氧化碳排放量标准，但对发展中国家和发达国家的排放量的限制采用了双重标准，为遏制全球变暖发达国家应尽更多的义务。《京都议定书》规定的各国二氧化碳的排放标准截止到201X
常用工具安全操作规程正式样本
文件编号：TP-AR-L9001 There Are Certain Management Mechanisms And Methods In The Management Of Organizations, And The Provisions Are Binding On The Personnel Within The Jurisdiction, Which Should Be Observed By Each Party. （示范文本） 编制：_______________ 审核：_______________ 单位：_______________ 常用工具安全操作规程 正式样本
常用工具安全操作规程正式样本 使用注意：该操作规程资料可用在组织/机构/单位管理上，形成一定的管理机制和管理原则、管理方法以及管理机构设置的规范，条款对管辖范围内人员具有约束力需各自遵守。材料内容可根据实际情况作相应修改，请在使用时认真阅读。 1、常用工具主要指：台钻、手电钻、砂轮、扳手、手锤、锉刀、手锯、凿子、冲子、螺丝刀、虎钳、千斤顶、刮刀等。 2、凡使用上述工具都必须熟知并认真执行本规程。 一、台钻 1、工作前应扎紧袖口，戴好帽子，女工必须将头发塞进帽子里。操作时严禁戴手套及围巾，手中不准拿回丝、花包布等物。 2、工作前先检查机器、工具、电气设备和安全防护装置是否完好。
3、工作时精力要集中，严禁手触动正在转动的任何部位。变换车速、测量工件以及装卸工件、钻头、夹具等都必须在机器停稳后进行。 4、工件必须垫稳、夹牢。必要时要在工件下面垫垫块。 5、将要钻透工件时，应减慢进刀速度。 6、钻孔时工作台面不准放置刀具、量具及其他物品。 7、松紧钻头必须用钻头钥匙，不准用锤子或其他东西敲打。 二、手电钻 1、认真执行台钻有关安全操作规程。 2、手电钻应使用三芯（单相）、四芯（三相）橡胶软线，出线头必须有胶圈固定，电线长度不得超过10米。
论共同但有区别责任原则
论共同但有区别责任原则 姓名：杜晓静 专业：国际法学 学号：09122326
论共同但有区别责任原则 摘要：共同但有区别责任原则勾勒出了发达国家和发展中国家在承担全球环境退化责任问题上的大致轮廓，在解决国际环境问题领域有着重要的作用。但该原则在实践的过程中也面临着重重挑战。本文介绍了该原则的历史发展，分析了它的内涵和合理性，最后结合其在国际环境法中的实践，对它的适用前景提出了设想。 关键词：共同但有区别责任国际环境法合理性
共同但有区别的责任原则是指由于地球生态系统的整体性和导致环境退化的各种不同因素，以及能力上的差异，各国对保护和改善全球环境负有共同的但是又有区别的责任。关于该项原则在国际环境法中的地位，一直存在着很大的争议。但毫无疑问，该原则在解决国际环境问题领域有着重要的作用，它勾勒出了发达国家和发展中国家在承担全球环境退化责任问题上的大致轮廓。 一、共同但有区别责任原则的历史发展 最开始，在国际环境保护领域只是强调“共同责任”。如1959年《南极条约》的序言指出：“……承认为了全人类的利益，南极应永远专为和平目的而使用，不应成为国际纷争的场所和对象”；又如1967年《外层空间条约》的序言指出：“……确认为了和平目的发展，探索利用外层空间，是全人类的共同利益”；1971年《拉姆萨尔湿地公约》的序言承认，“人类同其环境的相互依存关系”。[1]直到1972年《人类环境宣言》第一次将发达国家和发展中国家的环境问题区分了开来，被认为是该原则的萌芽阶段。它指出：“在发展国家中，环境问题大半是由于发展不足造成的。……因此，发展中国家必须致力于发展工作，牢记它们优先任务和保护及改善环境的必要，”“应筹集资金维护和改善环境，其中要照顾到发展中国家的情况和特殊性，照顾到它们的请求而额外的国际技术和财政援助的需要。”[2]文件虽然没有明确使用“区别责任”，但提到了要照顾发展中国家的情况和特殊性。 而1992年里约会议提出了“鉴于导致全球环境退化的各种不同因素，各国负有共同但有区别的责任”，从而确立了这一原则。1992年的《联合国气候框架公约》把发达国家置身于承担环境问题责任的前列，强调了这一原则。在《气候变化框架公约》第3条中明确提到了共同但有区别责任，“各缔约国应当在公平的基础上，并根据它们共同但又有区别的责任和能力，为人类当代和后代的利益保护气候系统。因此，发达国家缔约方应当率先对付气候变化及其不利影响。”这段话为“共同但有区别责任”指出了法理基础。 1997年《京都议定书》更是以“共同但有区别原则”为基础制定的，在国际实践中被誉为“共同而有区别原则”的典型代表。议定书明确规定了发达国家温室气体排放削减义务，发展中国家没有削减的义务。议定书中规定，在2008-2012年间，附件一国家应将其温室气体排放量从1990年的水平减少5%，并为这些国家规定了具体的减排指标；附件二国家是附件一国家中的工业化国家，除了承担减排义务外，还要为发展中国家提供新的额外资金，并向这些国家转移用于缓解和适应气候变化的先进技术；而发展中国家仅仅承担研究、报告、宣传、培训教育等一般义务，当然也可以自愿减排。[3] 共同但有区别责任原则被确立下来，并在国际环境法中被广泛的应用，它有着丰富而深刻的内涵。 二、共同但有区别责任原则的内涵 共同但有区别原则指，各国对保护全球负有共同的但是有区别的责任，包括共同责任和区别责任两方面。共同责任指国际社会的每个成员国不论大小、强弱，都有义务去保护地球环境。这一点很多国际环境条约上都得到了体现，如《生物多样性公约》确认了生物多样性保护是人类共同的关注事项。具体而言，共同责任可以从以下几个方面加以理解： (1)各国都有义务保护本国的环境。各国有责任采取有效的措施，通过制定一系列法规严格追究国内环境损害责任。 (2)各国有责任对该国管辖内的企业或个人的活动给他国造成环境损害承担
汽车修理工常用工具安全操作规程示范文本
汽车修理工常用工具安全操作规程示范文本 In The Actual Work Production Management, In Order To Ensure The Smooth Progress Of The Process, And Consider The Relationship Between Each Link, The Specific Requirements Of Each Link To Achieve Risk Control And Planning 某某管理中心 XX年XX月
汽车修理工常用工具安全操作规程示范 文本 使用指引：此操作规程资料应用在实际工作生产管理中为了保障过程顺利推进，同时考虑各个环节之间的关系，每个环节实现的具体要求而进行的风险控制与规划，并将危害降低到最小，文档经过下载可进行自定义修改，请根据实际需求进行调整与使用。 1、钳工台（桌） 1）、钳工台（桌）一般紧靠墙壁，人站在一面工作， 对面不准有人。如大型钳台对边有人工作时，钳台上必须 设置密度适当的安全网，钳台（桌）必须安装牢固，不得 作铁铮。 2）钳台（桌）上使用照明灯电压不得超过36伏。 3）钳台（桌）的上杂物要及时清理，工具和工件要放 在指定地点。 2、台虎钳 1）台虎钳上不要放置工具，以防滑下伤人。
2）使用转座台虎钳工作时，必须把固定螺丝扳紧。 3）台壶钳的丝杆、螺母要经常擦洗和加油，并保持经常清洁。如有损坏，不得使用。 4）钳口要保持完好，磨平时要及时修复以防工件滑脱。钳口紧固螺丝要经常检查，以防松动。不准使用已滑扣的螺丝。 5）用台虎钳夹持工件时，只许使用钳口最大行程的，不得用管子套在手柄上或用手锤捶击手柄。 6）工件必须放正夹紧，手柄朝下。 7）工件超出钳口部分太长，要加支承。装卸工件时，必须防止工件摔下伤人。 3、手锤（头） 1）手锤柄必须用硬质木料制成，大小长短要适宜。锤柄要有适当的斜度，捶头口必须加铁楔，以免工作时甩摔